The present invention relates to compounds that selectively inhibit the binding of ligands to the adhesion receptor, xcex14xcex21 integrin, also known as VLA-4. Compounds of the present invention are useful in the treatment and prevention of pathologies associated with VLA-4 mediated cell adhesion, such as inflammatory and autoimmune diseases, and tumor metastasis.
A primary feature of such pathologies as inflammation and autoimmune diseases is the accumulation of activated leukocytes in affected tissues. The process by which leukocytes transmigrate from the circulation at a site of inflammation involves a cascade of interactions that can be divided into four major steps: tethering and rolling, activation, firm adhesion, and transmigration (Springer, T., Ann. Rev. Physiol., 57:827 (1995)). Initially, leukocytes are lightly tethered to the endothelium and roll along its surface. This is followed by cell activation, mediated by soluble chemotactic stimuli, which initiates the development of a firmer bond between individual leukocytes and endothelial cells. The firm bond then results in the successful adhesion and transmigration of the leukocytes through endothelial cell junctions. The steps occur in series and each is essential for transmigration to occur. This also means that transmigration can be modulated at each step, thus providing a number of potential targets for pharmacological inhibition.
The receptors involved in leukocyte migration have, to a large extent, been characterized as belonging to particular cell adhesion molecule families (Carlos and Harlan, Blood, 84:2068 (1994)). The initial attachment and rolling step is mediated by a family of adhesion receptors referred to as selecting. Firm adhesion is mediated by interaction of leukocyte surface integrins with molecules of the immunoglobulin superfamily expressed on the surface of the endothelium. Both integrins and the immunoglobulin-type adhesion molecules are also primarily involved in leukocyte transmigration. After transmigration, the leukocytes rely on integrins to traverse through the extracellular matrix and remain at the site of inflammation.
Integrins are a large family of heterodimeric glycoproteins composed of two noncovalently associated subunits, xcex1 and xcex2 (Hynes, R., Cell, 69:11 (1992)). There are at least 16 different xcex1 subunits (xcex11-xcex19, xcex1L, xcex1M, xcex1D, xcex1X, xcex1E, xcex1IIb, xcex1v) and at least 9 different xcex2 (xcex21-xcex29) subunits. Integrins are divided into sub-families, based upon the xcex2 subunit. Leukocytes express a number of different integrins, including xcex14xcex21, xcex15xcex21, xcex16xcex21, xcex14xcex27, xcex1Lxcex22, xcex1Xxcex22, and xcex1Vxcex23.
xcex14xcex21 integrin, also known as very late antigen-4 (VLA-4) or CD49d/CD29, is expressed on monocytes, lymphocytes, eosinophils, and basophils, all of which are key effector cells in various inflammatory disorders (Helmer, M., Ann. Rev. Immunol., 8:365 (1990)). xcex14xcex21 integrin serves as a receptor for vascular cell adhesion molecule-1 (VCAM-1), as well as to the extracellular protein fibronectin (FN) (Elices et al., Cell, 60:577 (1990)). Anti-inflammatory effects and delayed disease progression have been demonstrated after in vivo monoclonal antibody blockade of the xcex14xcex21/VCAM-1 pathway (Lobb et al., J. Clin. Invest., 94:1722-28 (1994)). In a guinea pig model of pulmonary inflammation, anti-xcex14 inhibited both antigen-induced bronchial hyperreactivity and leukocyte recruitment in bronchoalveolar lavage fluid (Pretolani et al., J. Exp. Med., 180:795 (1994)). Antibodies to xcex14 or VCAM-1, prevented antigen-induced eosinophil infiltration of the mouse trachea (Nakajima et al., J. Exp. Med., 179:1145 (1994)). xcex14 or VCAM-1 monoclonal antibody treatment also delayed or prevented cutaneous delayed hypersensitivity response in mice and monkeys (Chisholm et al., Eur. J. Immunol., 23:682 (1993); Silber et al., J. Clin. Invest., 93:1554 (1993); cardiac allograft rejection in mice, accompanied by specific immunosuppression (Isobe et al., J. Immunol., 153:5810 (1994); graft-versus-host disease in mice after bone marrow transfer (Yang et al., Proc. Natl. Acad. Sci. USA, 90:10494, (1993); and experimental autoimmune encephalomyelitis in rats and mice (Yednock et al., Nature, 356:63 (1992); Baron et al., J. Exp. Med., 177:57 (1993)).
Rational drug design studies have produced soluble VCAM-Ig fusion protein containing the two N-terminal domains of human VCAM-1 fused to a human IgG1 constant region. In vivo administration of the fusion protein significantly delays the onset of adoptively transferred autoimmune diabetes in nonobese diabetic mice (Jakubowski et al., J. Immunol., 155:938 (1995)). Another approach has used three-dimensional crystallographic structures of VCAM-1 fragments to synthesize cyclic peptide antagonists that closely mimicked the xcex14 integrin binding loop in domain 1 of VCAM-1. Synthetic VCAM-1 peptide CQIDSPC, was able to inhibit the adhesion of VLA-4-expressing cells to purified VCAM-1 (Wang et al., Proc. Natl. Acad. Sci. USA, 92:5714 (1995)).
An additional strategy is to block the binding of xcex14xcex21 to its other counter receptor, that is, an alternatively spliced region of fibronectin containing the connecting segment-1 (CS-1) motif (E. A. Wayner, J. Cell. Biol., 116:489 (1992)). A synthetic CS-1 tetrapeptide (phenylacetic acid-Leu-Asp-Phe-d-Pro-amide) inhibited VLA-4-mediated lymphocyte adherence in vitro and reduced accelerated coronary arteriopathy in rabbit cardiac allografts (Molossi et al., J. Clin. Invest., 95:2601 (1995)). Each of these studies provide evidence that selective inhibition of xcex14xcex21/VCAM-1 mediated adhesion is a proven strategy in the treatment of autoimmune and allergic inflammatory diseases.
Moreover, while U.S. Pat. No. 5,821,231 and PCT Applications WO 96/22966, WO 97/03094, WO 98/04247 and WO 98/04913 describe compounds exhibiting VLA4 inhibitory activity in in vitro binding assays, none of the described compounds have exhibited efficacy in oral administration.
Accordingly, despite these advances, there remains a need for small, non-peptidic, specific inhibitors of VLA4 dependent cell adhesion that are orally bioavailable and that are suitable for the long-term treatment of chronic inflammatory diseases and other pathologies associated with leukocyte migration and adhesion.
The compounds of the present invention selcetively inhibit the binding of ligands to xcex14xcex21 and therefore, are useful for inhibition, prevention and suppression of VLA-4-mediated cell adhesion and the pathologies associated with that adhesion, such as, for example, inflammation, asthma, arthritis, diabetes, autoimmune responses, multiple sclerosis, psoriasis, transplantation rejection, and tumor metastasis.
In one embodiment, the present invention provides a compound represented by Formula I, or a salt thereof, 
wherein
W is chosen from aryl group, substituted aryl group, heteroaryl group and substituted heteroaryl group;
W1 is chosen from arylene group, substituted arylene group, heteroarylene group and substituted heteroarylene group;
A is chosen from xe2x95x90O, xe2x95x90S and xe2x95x90NH;
R is chosen from a direct bond, alkyenylene group and xe2x80x94(CH2)nxe2x80x94, wherein
n is chosen from 1 and 2;
X is chosen from xe2x80x94C(O)xe2x80x94, xe2x80x94CH2xe2x80x94 and S(O)2;
M is chosen from 
xe2x80x83wherein 
xe2x80x83is a divalent 4-, 5-, 6- or 7-membered heterocyclic moiety, wherein the nitrogen atom is the point of attachment to X;
R1, R2 and R3 are independently chosen from xe2x80x94H, xe2x80x94OH, xe2x80x94NH2, halogen atom, alkyl group, substituted alkyl group, aryl group, substituted aryl group, alkoxy group, substituted alkoxy group, monoalkylamino group, substituted monoalkylamino group, dialkylamino group, substituted dialkylamino group, cycloalkylamino group, substituted cycloalkylamino group, alkylsulfonylamino group, substituted alkylsulfonylamino group, alylsulfonylamino group, substituted arylsulfonylamino group, aryloxy group, substituted aryloxy group, heteroaryloxy group, substituted heteroaryloxy group, benzyloxy group and substituted benzyloxy group, or two of R1, R2 and R3 taken together may form a 3-, 4-, 5-, 6-, or 7-membered carbocyclic or heterocyclic residues optionally substituted with from 1 to 3 substituents chosen independently from xe2x80x94OH, halogen atom, xe2x80x94NH2, alkyl group, alkoxy group, aryl group, aryloxy group, alkylamino group, benzyloxy group and heteroaryl group;
R4 is chosen from xe2x80x94H and lower alkyl group;
Y is a direct bond or a divalent radical chosen from xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)NHxe2x80x94, alkenylene group, alkynylene group and xe2x80x94(CH2)kY2, wherein
k is chosen from 1, 2 and 3; and
Y2 is a direct bond or a divalent radical chosen from xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94S(O), xe2x80x94S(O)2xe2x80x94 and xe2x80x94NY3xe2x80x94, wherein
Y3 is chosen from xe2x80x94H and lower alkyl group;
Z is chosen from arylene group, substituted arylene group, heterocyclylene group, substituted heterocyclylene group, cycloalkylene group and substituted cycloalkylene group;
A1 is a direct bond or a divalent radical chosen from alkenylene group, alkynylene group, xe2x80x94(CH2)txe2x80x94 and xe2x80x94O(CH2)v, wherein
t is chosen from 1, 2 and 3; and
v is chosen from 0, 1, 2, and 3; and
R5 is chosen from xe2x80x94OH, lower alkoxy group, xe2x80x94N(H)OH, 
xe2x80x83wherein 
xe2x80x83is a divalent 4-, 5-, 6- or 7-membered heterocyclic moiety, wherein the nitrogen atom is the point of attachment to X;
R6 and R7 are independently chosen from xe2x80x94H, xe2x80x94OH, halogen atom alkyl group and alkoxy group;
Y1 is a divalent radical chosen from xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)2xe2x80x94 and xe2x80x94NY4xe2x80x94, wherein
Y4 is chosen from xe2x80x94H and lower alkyl group;
Z1 is a divalent radical chosen from arylene group, substituted arylene group, heterocyclylene group, substituted heterocyclylene group, cycloalkylene group and substituted cycloalkylene group;
A2 is a direct bond or a divalent radical chosen from alkenylene group, alkynylene group and xe2x80x94(CH2)e wherein
e is chosen from 1, 2 and 3; and
R8 is chosen from xe2x80x94OH, lower alkoxy group, xe2x80x94N(H)OH, 
xe2x80x83wherein
L is 
xe2x80x83wherein 
xe2x80x83is a divalent 4-, 5-, 6- or 7-membered heterocyclic moiety, optionally substituted with from 1 to 3 substitutents chosen independently from alkyl group, alkoxy group, hydroxyalkyl group, xe2x80x94OH, benzyloxy group, xe2x80x94NH2, halogen atom, aryl group and heteroaryl group, said moiety may be fused to 1 or 2 additional carbocyclic or heterocyclic residues optionally substituted with from 1 to 3 substitutents chosen independently from alkyl group, aryloxy group, alkoxy group, hydroxyalkyl group, xe2x80x94OH, benzyloxy group, xe2x80x94NH2, halogen atom, aryl group and heteroaryl group;
m and q are independently chosen from 0, 1, 2 and 3;
X1 is chosen from xe2x80x94CHxe2x95x90 and xe2x80x94Nxe2x95x90;
R9 is chosen from xe2x80x94H and lower alkyl group;
R10 is chosen from xe2x80x94COOH, lower alkoxycarbonyl group, 
Z2 is chosen from xe2x80x94F, COOH and lower alkoxycarbonyl group; and 
xe2x80x83wherein
R11 is chosen from xe2x80x94Oxe2x80x94, 
xe2x80x83and xe2x80x94NR12xe2x80x94 wherein
R12 is chosen from xe2x80x94H alkyl group, substituted alkyl group, cycloalkyl group, substituted cycloalkyl group, aryl group, substituted aryl group, benzyl group, substituted benzyl group, lower alkenyl group, substituted lower alkenyl group and lower alkynyl group
the left hand bond is the point of attachment to xe2x80x94Xxe2x80x94 and the right hand bond is the point of attachment to xe2x80x94Z3;
Z3 is chosen from a direct bond, a divalent aliphatic hydrocarbon moiety having 1 to 12 carbon atoms, wherein
one or more carbon atoms may be replaced with xe2x80x94Oxe2x80x94 or xe2x80x94NR13xe2x80x94 wherein
R13 is chosen from xe2x80x94H and lower alkyl group, and one or more hydrogen atoms attached to an aliphatic carbon atom may be replaced with lower alkyl group; and 
xe2x80x83wherein
x is chosen from 0 and 1;
y is chosen from 1, 2, and 3; and
R14 is chosen from xe2x80x94H, xe2x80x94OH and halogen atom, 
xe2x80x83when
R11 is xe2x80x94NR12, 
xe2x80x83wherein 
xe2x80x83wherein
Z4 is chosen from 
xe2x80x83wherein
R14a is chosen from xe2x80x94H, xe2x80x94OH, lower alkyl group and halogen atom; 
wherein the left hand bond is the point of attachment to R11 and the right hand bond is the point of attachment to Q2;
Q2 is a divalent radical chosen from arylene group, substituted arylene group, heterocyclylene group, substituted heterocyclylene group, cycloalkylene group, substituted cycloalkylene group, 
xe2x80x83wherein R15 and R16 are independently chosen from xe2x80x94H, halogen atom and lower alkyl group; and 
xe2x80x83wherein R17 and R18 are independently chosen from xe2x80x94H, lower alkyl group, substituted lower alkyl group and lower alkenyl group; and
L1 is chosen from xe2x80x94COOH and xe2x80x94COOR19 wherein
R19 is a lower alkyl group.
In a preferred embodiment of Formula I, M is 
In this embodiment, more preferred compounds are those wherein A is xe2x95x90O, R is xe2x80x94(CH2)nxe2x80x94 and X is xe2x80x94C(O)xe2x80x94 Y is preferably chosen from alkenylene group, alkynylene group, xe2x80x94(CH2)kY2, xe2x80x94CH2S(O)xe2x80x94 and xe2x80x94CH2Oxe2x80x94, and more preferably, Y is xe2x80x94CH2Oxe2x80x94.
Preferred compounds of this embodiment are those wherein W is unsubstituted phenyl group or phenyl group having one or two substituents chosen from lower alkyl group and halogen atom at the ortho positions thereof. W1 is preferably unsubstituted phenylene group or phenylene group having a substituent chosen from methoxy group, lower alkyl group and halogen atom at the ortho position to xe2x80x94NHxe2x80x94.
In preferred compounds of this embodiment, A is preferably xe2x95x90O and A1 is a direct bond or xe2x80x94(CH2)txe2x80x94. More preferred compounds are those wherein A1 is a direct bond and R5 is xe2x80x94OH.
Preferred compounds of Formula I, wherein M is 
and A is xe2x95x90O are represented in Table 1. With respect to the representation of xe2x80x94W1, the lower bond is the point of attachment to xe2x80x94NHxe2x80x94 and the upper bond is the point of attachment to xe2x80x94Rxe2x80x94. The entry entitled xe2x80x94Rxe2x80x94R5 depicts that portion of the particular compound represented by 
In another preferred embodiment of Formula I, M is 
In this embodiment, more preferred compounds are those wherein A is xe2x95x90O, R is xe2x80x94(CH2)nxe2x80x94 and X is xe2x80x94C(O)xe2x80x94. Y is preferably chosen from xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)2xe2x80x94 and xe2x80x94NY4, and more preferably, is xe2x80x94Oxe2x80x94.
Preferred compounds of this embodiment are those wherein W is unsubstituted phenyl group or phenyl group having one or two substituents chosen from lower allyl group and halogen atom at the ortho positions thereof. W1 is preferably unsubstituted phenylene group or phenylene group having a substituent chosen from methoxy group, lower alkyl group and halogen atom at the ortho position to xe2x80x94NHxe2x80x94.
In this embodiment of Formula I, A is preferably xe2x95x90O and A2 is a direct bond or xe2x80x94(CH2)exe2x80x94. More preferred compounds are those wherein A2 is a direct bond and R8 is xe2x80x94OH.
Preferred compounds of Formula I, wherein M is 
and A is xe2x95x90O, are represented in Table 2. With respect to the representation of xe2x80x94W1, the lower bond is the point of attachment to xe2x80x94NHxe2x80x94 and the upper bond is the point of attachment to xe2x80x94Rxe2x80x94. The entry entitled xe2x80x94Rxe2x80x94R8 depicts that portion of the particular compound represented by 
In another preferred embodiment of Formula I, M is 
In this embodiment, preferred compounds are those wherein A is xe2x95x90O, R10 is xe2x80x94CO2H and q is 0 or 1. More preferred are compounds wherein R10 is xe2x80x94CO2H, q is 0 or 1, most preferably 0, and m is 2.
When A is xe2x95x90O, L is preferably chosen from 
More preferably, L is chosen from 
Most preferably, L is chosen from 
Preferred compounds of Formula I, wherein R is xe2x80x94CH2xe2x80x94 and X is xe2x95x90O, are those wherein W is unsubstituted phenyl group or phenyl group having one or two substituents chosen from lower alkyl group and halogen atom at the ortho positions thereof. W1 is preferably unsubstituted phenylene group or phenylene group having a substituent chosen from methoxy group, lower alkyl group and halogen atom at the ortho position to xe2x80x94NHxe2x80x94.
Preferred compounds of Formula I, wherein M is 
A is xe2x95x90O, R is xe2x80x94CH2xe2x80x94 and X is xe2x95x90O, are represented in Table 3. With respect to the representation of xe2x80x94W1, the lower bond is the point of attachment to xe2x80x94NHxe2x80x94 and the upper bond is the point of attachment to xe2x80x94Rxe2x80x94. The entry entitled xe2x80x94Nxe2x80x94Z2 depicts that portion of the particular compound represented by 
Yet another preferred embodiment of Formula I includes compounds wherein M is 
Preferably, A is xe2x95x90O, R is xe2x80x94CH2xe2x80x94 and X is xe2x95x90O. Preferably W is unsubstituted phenyl group or phenyl group having one or two substituents chosen from lower alkyl group and halogen atom at the ortho positions thereof. W1 is preferably unsubstituted phenylene group or phenylene group having a substituent chosen from methoxy group, lower alkyl group and halogen atom at the ortho position to xe2x80x94NHxe2x80x94.
In compounds wherein Q2 is 
and Z3 is a divalent aliphatic hydrocarbon moiety, preferred compounds are those wherein R11 is 
or xe2x80x94NR12, more preferably NR12, wherein R12 is chosen from xe2x80x94H lower alkyl group and substituted lower alkyl group, most preferably dihydroxy lower alkyl group. Preferred choices for Z3 is a divalent aliphatic hydrocarbon moiety having 4, 5 or 6 carbon atoms. A preferred choice for W1 is phenylene group having a substituent chosen from methoxy group, lower alkyl group and halogen atom at the ortho position to xe2x80x94NHxe2x80x94.
In compounds wherein Q2 is 
and Z3 is 
R11 is preferably xe2x80x94NR12xe2x80x94. In these compounds, x and y are preferably 1. Preferred choices for R14 include xe2x80x94H. xe2x80x94OH and xe2x80x94F. A preferred choice for W1 is phenylene group having a substituent chosen from methoxy group, lower alkyl group and halogen atom at the ortho position to xe2x80x94NHxe2x80x94.
In compounds wherein Q2 is 
and Z3 is 
R11 is preferably chosen from xe2x80x94Oxe2x80x94 and xe2x80x94NR12xe2x80x94, preferably wherein R12 is chosen from xe2x80x94H and lower alkyl group. Preferably, R17 and R18 are each xe2x80x94H. A preferred choice for W1 is phenylene group having a substituent chosen from methoxy group, lower alkyl group and halogen atom at the ortho position to xe2x80x94NHxe2x80x94.
In compounds wherein Q2 is 
and Z3 is 
R11 is preferably xe2x80x94NR12, wherein R12 is preferably lower alkyl group. Preferred compounds of this embodiment also include those wherein at least one of R17 and R18 is lower alkyl group or substituted lower alkyl group.
In compounds wherein Q2 is 
and Z3 is 
R11 is preferably xe2x80x94NHxe2x80x94 and R17 and R18 are each preferably xe2x80x94H. A preferred choice for W1 is phenylene group having a substituent chosen from methoxy group, lower alkyl group and halogen atom at the ortho position to xe2x80x94NHxe2x80x94.
In compounds wherein Q2 is chosen from aryl group, substituted aryl group and 
and more preferably from phenyl group and phenyl group substituted at the point of attachment to Z3, Z3 is preferably a divalent aliphatic hydrocarbon moiety.
Yet another embodiment of the invention is a compound represented by Formula II, 
wherein the substituents W, W1, R11 and Z3 are defined as in Formula I, and L3 is chosen from 
wherein R20 is preferably chosen from xe2x80x94H and lower alkyl, 
Still another embodiment of the invention is a compound represented by Formula III, 
wherein the substituents W, W1, and R11 are defined as in Formula I, and d is chosen from 0 and 1, and f is chosen from 1 and 2.
Preferred compounds of Formula I, wherein M is 
A is xe2x95x90O, R is xe2x80x94CH2xe2x80x94 and X is xe2x95x90O, are represented in Table 4. With respect to the representation of xe2x80x94W1, the lower bond is the point of attachment to xe2x80x94NHxe2x80x94 and the upper bond is the point of attachment to xe2x80x94Rxe2x80x94. The entry entitled xe2x80x94R11xe2x80x94L1 depicts that portion of the particular compound represented by 
The principles of the present invention also encompass prodrugs in the scope of Formula I, and compounds representative thereof include those wherein R5 or R8 is a lower alkoxy group, and those wherein R10 or R19 is a lower alkoxycarbonyl group.
The principles of the present invention also provide a method for inhibiting cell adhesion, and in particular, VLA-4 mediated cell adhesion at xcex14xcex21 receptor sites in a mammal, wherein the method comprises administering an effective amount of a compound represented by Formula I. As used herein, inhibiting cell adhesion is intended to include inhibiting, suppressing and preventing VLA-4 mediated cell adhesion-associated conditions, including but not limited to, inflammation and cell adhesion-associated immune or autoimmune responses.
The principles of the present invention therefore also provide a method of treating a condition associated with VLA4 mediated cell adhesion, wherein the method comprises administering to a mammal in need of such treatment, an effective amount of a compound represented by Formula I. Such conditions include for example, but are not limited to, inflammatory and autoimmune responses, diabetes, asthma, arthritis, psoriasis, multiple sclerosis, inflammatory bowel disease, transplantation rejection, and tumor metastasis. As used herein, xe2x80x9ctreatmentxe2x80x9d of a mammal is intended to include prophylaxis as well.
The compounds of the present invention may be administered as a monotherapy, or in combination with antiinflammatory or immunosuppressive agents. Such combination therapies can involve the administration of the various pharmaceuticals as a single dosage form or as multiple dosage forms administered at the same time or at different times.
Any suitable route of administration may be employed for providing a patient with an effective amount of a compound of the present invention. Suitable routes of administration may include, for example, oral, rectal, nasal, buccal, parenteral (such as, intravenous, intrathecal, subcutaneous, intramuscular, intrasternal, intrahepatic, intralesional, intracranial, intra-articular, and intra novial), transdermal (such as, for example, patches), and the like. Due to their ease of administration, oral dosage forms, such as, for example, tablets, troches, dispersions, suspensions, solutions, capsules, soft gelatin capsules, and the like, may be preferred. Administration may also be by controlled or sustained release means and delivery devices. Methods for the preparation of such dosage forms are well known in the art.
Pharmaceutical compositions incorporating compounds of the present invention may include excipients, a pharmaceutically acceptable carrier, in addition to other therapeutic ingredients. Excipients such as starches, sugars, microcrystalline cellulose, diluents, lubricants, binders, coloring agents, flavoring agents, granulating agents, disintegrating agents, and the like may be appropriate depending upon the route of administration. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.
The compounds of the present invention may be used in the form of pharmaceutically acceptable salts derived from inorganic or organic bases. Suitable pharmaceutically acceptable base addition salts include, but are not limited to, ammonium salts, alkali metal salts, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, organic salts made from chloroprocaine, choline, N,Nxe2x80x2-dibenzylethylenediamine, dicyclohexylamine, diethanolamine, ethylenediamine, lysine, meglumine (N-methylglucamine) and procaine, as well as salts with amino acids, such as arginine, lysine, and so forth.
Where the compounds of the invention have a basic moiety, such as an amino group, the compounds may be used in the form of pharmaceutically acceptable non-toxic organic or inorganic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, methanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, lactic, maleic, malic, mandelic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acids, and the like. Particularly preferred are citric, hydrochloric, maleic, fumaric, phosphoric, sulfuric, tartaric and p-toluenesulfonic acids. Compounds of the invention may also be in the form of hydrates.
The following terms and abbreviations have the indicated meaning throughout this disclosure.
xe2x80x9cAlkyl groupxe2x80x9d is intended to include linear or branched hydrocarbon radicals and combinations thereof of 1 to 20 carbons. xe2x80x9cLower alkyl groupxe2x80x9d means alkyl groups of from 1 to about 10, preferably from 1 to about 8, and more preferably, from 1 to about 6 carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl pentyl, iso-amyl hexyl, octyl groups and the like.
xe2x80x9cAlkylene groupxe2x80x9d means a divalent radical formed by removing a hydrogen atom from an xe2x80x9callyl group.xe2x80x9d
xe2x80x9cAryl groupxe2x80x9d means a radical formed from an aromatic hydrocarbon ring of 4 to about 16 carbon atoms, preferably of 6 to about 12 carbon atoms, and more preferably of 6 to about 10 carbon atoms. The rings may optionally be substituted with 1-3 substituents selected from alkyl, halogen, hydroxy, alkoxy, aryloxy, haloalkyl phenyl and heteroaryl. Examples of aryl groups are phenyl, biphenyl, 3,4-dichlorophenyl and naphthyl.
xe2x80x9cArylene groupxe2x80x9d means a divalent radical formed by removing a hydrogen atom from an xe2x80x9caryl group.xe2x80x9d
xe2x80x9cArylalkyl groupxe2x80x9d denotes a structure comprising an alkyl attached to an aryl ring. Examples include benzyl, phenethyl, 4-chlorobenzyl, and the like.
xe2x80x9cCycloalkyl groupxe2x80x9d refers to a saturated hydrocarbon ring radical of from 3 to 12 carbon atoms, and preferably from 3 to 8 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, adamantyl, myrtanyl groups and the like. xe2x80x9cLower cycloalkyl groupxe2x80x9d refers to cycloalkyl of 3 to 6 carbons.
xe2x80x9cCycloalkylene groupxe2x80x9d means a divalent radical formed by removing a hydrogen atom from a xe2x80x9ccycloalkyl group.xe2x80x9d
xe2x80x9cDivalent C1 to C20 aliphatic hydrocarbon moietyxe2x80x9d includes alkylene, cycloalkylene, alkenylene, alkynylene groups and combinations thereof Examples include ethylene, propylene, propynylene, 2,4-heptadienylene groups and the like.
xe2x80x9cHeterocyclyl groupxe2x80x9d refers to a cyclic radical having from 1 to 6 carbon atoms, preferably 3 to 6 carbon atoms, and from 1 to 4 heteroatoms chosen from O, N and S. Examples include: pyrrolyl, pyridinyl, pyrazolyl triazolyl pyrimidinyl, pyridazinyl, oxazolyl, thiazolyl, imidazolyl, indolyl, thienyl, furyl, azetidiyl, tetrazolyl, 2-pyrrolinyl 3-pyrrolinyl, pyrrolindinyl, 1,3-dioxolanyl, imidazolinyl, imidazolidinyl, pyrazolinyl pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4H-pyranyl piperidinyl 1,4-dithianyl, morpholinyl thiomorpholinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl, 1,2,5-trithianyl, benzo(b)thiophenyl, benzimidazolyl, quinolinyl groups and the like.
xe2x80x9cHeterocyclylene groupxe2x80x9d means a radical formed by removing a hydrogen atom from a xe2x80x9cheterocyclyl group.xe2x80x9d
xe2x80x9cHeteroaryl groupxe2x80x9d refers to an aromatic cyclic radical having from 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms, and from 1 to 4 heteroatoms chosen from O, N and S; or a bicyclic 9- or 10-membered heteroaromatic ring system containing 1-4 heteroatoms selected from O, N and S. The methine H atoms of a heterocyclyl or heteroaryl structure may be optionally substituted with alkyl, alkoxy or halogen. Examples include: imidazolyl, pyridyl, indolyl, thienyl, benzopyranyl, thiazolyl, furyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, pyrimidinyl, pyrazinyl tetrazolyl, pyrazolyl groups and the like.
xe2x80x9cHeteroarylene groupxe2x80x9d means a divalent radical formed by removing a hydrogen atom from a xe2x80x9cheteroaryl group.xe2x80x9d
xe2x80x9cAlkoxy groupxe2x80x9d means a straight, branched or cyclic hydrocarbon configuration and combinations thereof, including from 1 to 20 carbon atoms, preferably from 1 to 8 carbon atoms, more preferably from 1 to 4 carbon atoms, and an oxygen atom at the point of attachment. Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, cyclopropoxy, cyclohexyloxy groups and the like. xe2x80x9cLower alkoxy groupxe2x80x9d refers to alkoxy groups having from 1 to 4 carbon atoms.
xe2x80x9cAlkenyl groupxe2x80x9d refers to an unsaturated acyclic hydrocarbon radical in so much as it contains at least one double bond. xe2x80x9cLower alkenyl groupxe2x80x9d refers to such radicals containing from 2 to 10 carbon atoms, preferably from 2 to 8 carbon atoms and more preferably from 2 to 6 carbon atoms. Examples of suitable alkenyl radicals include propenyl, buten-1-yl, isobutenyl, penten-1-yl, 2-methylbuten-1-yl, 3-methylbuten-1-yl, hexen-1-yl, hepten-1-yl, and octen-1-yl groups and the like.
xe2x80x9cAlkenylene groupxe2x80x9d means a divalent radical formed by removing a hydrogen atom from an xe2x80x9calkenyl group.xe2x80x9d
xe2x80x9cAlkynyl groupxe2x80x9d refers to an unsaturated acyclic hydrocarbon radical containing at least one triple bond. Examples include ethynyl, propynyl groups, and the like.
xe2x80x9cAlkynylene groupxe2x80x9d means a divalent radical formed by removing a hydrogen atom from an alkynyl group.xe2x80x9d
xe2x80x9cSubstituted alkyl groupxe2x80x9d means a linear or branched alkyl group wherein at leas one hydrogen atom attached to an aliphatic carbon is replaced with a substituent such as alkyl, amino, alkoxy, hydroxy, aryl, cyano, carboxy, alkoxycarbonyl, monoakylamino, alkyloxy, cyanoalkyl, cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, carboxyalkyl, alkoxycarbonylalkyl, haloalkyl, acylamino, dialkylamino, cyclicamino groups, halogen atom and nitro. Examples of such substituent groups include methyl, isopropyl, methoxy, ethoxy, propoxy amino, methylamino, phenyl, naphthyl groups, chlorine, fluorine and the like.
xe2x80x9cSubstituted alkylene groupxe2x80x9d means a linear or branched alkylene group wherein at least one hydrogen atom attached to an aliphatic carbon is replaced with a substituent such as alkyl, amino, alkoxy, hydroxy, aryl, cyano, carboxy, alkoxycarbonyl, monoalkylamino, alkyloxy, cyanoalkyl, cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, carboxyalkyl, alkoxycarbonylalkyl, haloalkyl, acylamino, dialkylamino, cyclicamino groups, halogen atom and nitro.
xe2x80x9cSubstituted cycloalkyl groupxe2x80x9d means a cycloalkyl group wherein at least one hydrogen atom attached to a ring carbon atom is replaced with a substituent such as alkyl, amino, alkoxy, hydroxy, aryl, cyano, carboxy, alkoxycarbonyl, monoalkylamino, alkyloxy, cyanoalkyl, cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, carboxyalkyl, alkoxycarbonylalkyl, haloalkyl, acylamino, dialkylamino, cyclicamino groups, halogen atom and nitro.
xe2x80x9cSubstituted cycloalkyene groupxe2x80x9d means a cycloalkylene group wherein at least one hydrogen atom attached to a ring carbon is replaced with a substituent such as alkyl, amino, alkoxy, hydroxy, aryl, cyano, carboxy, alkoxycarbonyl, monoalkylamino, alkyloxy, cyanoalkyl, cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, carboxyalkyl, alkoxycarbonylalkyl, haloalkyl, acylamino, dialkylamino, cyclicamino groups, halogen atom and nitro.
xe2x80x9cSubstituted aryl groupxe2x80x9d means an aryl group wherein at least one methine hydrogen atom attached to an aromatic carbon is replaced with a substituent such as alkyl, amino, alkoxy, hydroxy, aryl cyano, carboxy, alkoxycarbonyl, monoalkylamino, alkyloxy, cyanoalkyl, cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, carboxyalkyl, alkoxycarbonylalkyl, haloalkyl, acylamino, dialkylamino, cyclicamino groups, halogen atom and nitro.
Substituted arylene groupxe2x80x9d means an arylene group wherein at least one hydrogen atom attached to an aromatic carbon is replaced with a substituent such as alkyl, amino, alkoxy, hydroxy, aryl, cyano, carboxy, alkoxycarbonyl, monoalkylamino, alkyloxy, cyanoalkyl cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, carboxyalkyl, alkoxycarbonylalkyl, haloalkyl, acylamino, dialkylamino, cyclicamino groups, halogen atom and nitro.
xe2x80x9cSubstituted heteroaryl groupxe2x80x9d or xe2x80x9csubstituted heterocyclyl groupxe2x80x9d means a heteroaryl or heterocyclyl group wherein at least one hydrogen atom attached to a ring thereof is replaced with a substituent such as alkyl, amino, alkoxy, hydroxy, aryl cyano, carboxy, alkoxycarbonyl, monoalkylamino, alkyloxy, cyanoalkyl, cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, carboxyalkyl, alkoxycarbonylalkyl, haloalkyl, acylamino, dialkylamino, cyclicamino groups, halogen atom and nitro.
xe2x80x9cSubstituted heteroarylene groupxe2x80x9d or xe2x80x9csubstituted heterocyclylene groupxe2x80x9d means a heteroarylene or heterocyclylene group wherein at least one hydrogen atom attached to a ring thereof is replaced with a substituent such as alkyl, amino, alkoxy, hydroxy, aryl, cyano, carboxy, alkoxycarbonyl, monoalkylamino, alkyloxy, cyanoalkyl, cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, carboxyalkyl, alkoxycarbonylalkyl, haloalkyl, acylamino, dialkylamino, cyclicamino groups, halogen atom and nitro.
xe2x80x9cSubstituted arylalkyl groupxe2x80x9d means an arylalkyl having one or more substituents such as alkyl, amino, alkoxy, hydroxy, aryl, cyano, carboxy, alkoxycarbonyl, monoalkylamino, alkyloxy, cyanoalkyl, cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, carboxyalkyl, haloalkyl, alkoxycarbonylalkyl, acylamino, dialkylamino, cyclicamino groups, halogen atom and nitro.
xe2x80x9cHalogenxe2x80x9d is intended to include for example, F, Cl, Br and I.
The term xe2x80x9cprodrugxe2x80x9d refers to a chemical compound that is converted to an active agent by metabolic processes in vivo. [See, e.g., N. Boder and J. J. Kaminski, Ann. Rep. Med. Chem. 22:303 (1987) and H. Bundgarrd, Adv. Drug Delivery Rev., 3:39 (1989)]. The use of prodrug precursors of compounds of the present invention in any of the methods described herein is contemplated and is intended to be within the scope of the invention.
Terminology related to xe2x80x9cprotected,xe2x80x9d xe2x80x9cprotectingxe2x80x9d and/or xe2x80x9cdeprotectingxe2x80x9d functionalities is used throughout this application. Such terminology is well understood by persons of skill in the art and is used in the context of processes which involve sequential treatment with a series of reagents. In this context, a protecting group refers to a group which is used to mask a functionality during a process step in which it would otherwise react, but in which reaction is undesirable. The protecting group prevents reaction at that step, but may be subsequently removed to expose the original functionality. The removal or xe2x80x9cdeprotectionxe2x80x9d occurs after the completion of the reaction or reactions in which the functionality would interfere. Thus, when a sequence of reagents is specified, as it is in the processes of the invention, the person of ordinary skill can readily envision those groups that would be suitable as xe2x80x9cprotecting groupsxe2x80x9d for the functionalities involved.
In the case of the present invention, the functionalities that must be protected are amines. Suitable groups for that purpose are discussed in standard textbooks in the field of chemistry, such as Protective Groups in Organic Synthesis by T. W. Greene [John Wiley and Sons, New York, 1991], which is incorporated herein by reference. Particular attention is drawn to the chapter entitled xe2x80x9cProtection for the Amino Groupxe2x80x9d (pages 309-405). Preferred protecting groups include BOC and Fmoc. Exemplary methods for protecting and deprotecting with these groups are found in Greene and Wuts on pages 318 and 327.
The materials upon which the syntheses described herein are performed are referred to as solid supports, beads, and resins. These terms are intended to include: (a) beads, pellets, disks, fibers, gels, or particles such as cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-liked with divinylbenzene and optionally grafted with polyethylene glycol, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N,Nxe2x80x2-bis-acryloyl ethylene diamine, glass particles coated with hydrophobic polymer, etc, i.e., material having a rigid or semi-rigid surface; and (b) soluble supports such as polyethylene glycol or low molecular weight, non-cross-linked polystyrene. The solid supports may, and usually do, have functional groups such as amino, hydroxy, carboxy, or halo groups; where amino groups are the most common.
Tentagel(trademark) NH2 (Rapp Polymere, Tubingen, Germany) is a preferred amine functionalized polyethylene glycol-grafted polystyrene resin. Tentagel(trademark)-S-PHB resin has a para-hydroxy benzyl linker which can be cleaved by the use of 90% trifluoroacetic acid in dichloromethane. Techniques for functionalizing the surface of solid phases are well known in the art. Attachment of lysine to the amino groups on a bead (to increase the number of available sites) and subsequent attachment of linkers as well as further steps in a typical combinatorial synthesis are described, for example, in PCT application WO95/30642, the disclosure of which is incorporated herein by reference. In the synthesis described in WO95/30642, the linker is a photolytically cleavable linker, but the general principles of the use of a linker are well illustrated.
Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisometric forms which may be defined in terms of absolute stereochemistry as (R)- or (S)-, or as (D)- or (L)- for amino acids. The present invention is meant to include all such possible diastereomers as well as their racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended to include both (E)- and (Z)-geometric isomers. Likewise, all tautomeric forms are intended to be included. The configuration of any carbon-carbon double bond appearing herein is selected for convenience only and is not intended to designate a particular configuration; thus a carbon-carbon double bond depicted arbitrarily herein as trans may be cis, trans, or a mixture of the two in any proportion.
In view of the above definitions, other chemical terms used throughout this application can be easily understood by those of skill in the art. Terms may be used alone or in any combination thereof. The preferred and more preferred chain lengths of the radicals apply to all such combinations.
The compounds of the present invention have demonstrated utility as selective inhibitors at VLA-4 receptors. The inhibitory concentration (IC50) and the VLA-4 selectivity of test compounds for an xcex14xcex21 receptor using in vitro assays are determined in direct binding assays and competitive assays with other integrin receptors such as xcex22 (LFA-1 and Mac-1), xcex23 (GPIIb/IIIa and xcex1vxcex23) and xcex21 (xcex14xcex27). Compounds of the present invention have Ki values  less than 1 xcexcM. Preferred compounds of the invention are those having Ki values  less than 300 nM, more preferably  less than 100 nM, even more preferably  less than 50 nM, and most preferably,  less than 12 nM.
Examples of preferred compounds having a Ki value  less than 50 nM are shown below. These examples are provided by way of illustration only, and are not intended to limit the invention thereto. 
A direct binding assay was used to quantify the inhibitory activity of the compounds. In this assay, VLA-4-expressing cells were seeded in a 96-well microtiter plate. The cells were allowed to grow for 2 days until confluent. Various concentrations of the test compound were added together with 2 nM of the europium-labeled, VCAM-IgG fusion protein. The cells were allowed to incubate at room temperature in the microwells for at least 30 minutes. Following incubation, the microwells were emptied and washed. The amount of europium-labeled VCAM-IgG fusion protein bound was determined by time-resolved fluorescence measurement. Inhibition of binding was determined by quantifying the fluorescence bound to the plate for each of the various concentrations of test compound, as well as for controls containing no test compound.
The VLA4-expressing cells used in this assay was a CHO cell line stably transfected with the cDNA of the human xcex14 and xcex21 subunits. Construction and maintenance of the cell line are described in the assay procedures. A VCAM IgG fusion protein containing the one to seven immunoglobulin domains of human VCAM-1 (D1D7) attached above the hinge region of an IgG1 molecule was labeled with europium chelates. The preparation and labeling of the fusion protein are described in the assay procedures.
The cell adhesion inhibitory activity of the test compound was determined by blocking the Jurkat cell attachment to the D1D7-VCAM IgG fusion protein Jurkat cell is a human lymphocytic cell line expressing VLA4 on cell surface. In this assay, each of the 96-well microtiter wells was coated with 75 ng of the VCAM IgG fusion protein. The wells were then blocked by the addition of 1% bovine serum albumin to remove nonspecific adhesive sites. Varying concentrations of the test compound were added together with the calcein-labeled Jurkat cells. The cells were allowed to adhere to the VCAM coated wells at room temperature for 1 hour in the dark. Following incubation, the plate was washed by immersing face down into a container filled with phosphate buffered saline. The wells were blotted dry on paper towel. Quantitation of the adhered cell was determined by fluorescence measurement. Decreased fluorescence indicated inhibition of cell adhesion by the test compound.
Specificity for xcex14xcex21 of each test compound among other integrin receptors, namely, xcex22 (LFA-1 and Mac-1), xcex23 (GPIIb/IIIa and xcex1vxcex23), xcex21 (xcex15xcex21) and xcex27 (xcex14xcex27) was examined. LFA-1 binds to ICAM-1 and mediates the emigration of leukocytes into inflammatory sites. Mac-1 binds to a number of ligands, including ICAM-1 and fibrinogen, and plays an important role in neutrophil phagocytosis and oxygen free radical generation. GPIIb/IIIa on platelet surface binds to fibrinogen in plasma and induces platelet aggregation. xcex1vxcex23 binds to a number of extracellular matrix proteins, including vitronectin and mediates cell migration and prevents cell apoptosis. xcex14xcex27 shares the same ligands as VLA-4 (VCAM-1, MAdCAM-1, and fibronectin), but with different preference. This receptor is expressed on lymphoid cells and is involved in lymphocyte migration to mucosal tissues.
Assays of LFA-1, Mac-1, GPIIb/IIIa and xcex1vxcex23 involved coating the purified receptor on a 96-well microtiter plate. The specific ligands for these receptors were labeled with europium chelates. In the assays of LFA-1 and Mac-1, an ICAM-1 IgG fusion protein containing the one to five immunoglobulin domains of human ICAM-1 (D1D5) attached above the hinge region of an IgG1 molecule, was used. In the assays of GPIIb/IIIa and xcex1vxcex23, europium-labeled fibrinogen and vitronectin, respectively, was used. The purified receptors were allowed to incubate in the wells with various concentrations of test compound, in the presence of europium-labeled ligands. Following incubation, the wells are emptied and washed. The amount of europium-labeled ligand bound was determined by time-resolved fluorescence measurement. Assay of xcex14xcex27 is similar to the adhesion inhibition assay of VLA-4 described above, and uses the xcex14xcex27-expressing cell, RPMI-8886. A MAdCAM-1 IgG fusion protein containing the one and two immunoglobulin domains of human MAdCAM-1 and mucin-like repeat domain, is used as the corresponding ligand for xcex14xcex27.
Eu3+-VCAM-1 IgG binding to CHO/VLA-4 cells may be determined as follows. 4B4 cells (CHO/VLA-4 cells) are distributed into each well of a 96-well microtiter plate at 3xc3x97104/well. The plate is incubated at 37xc2x0 C., 5% CO2 for 48 hours and then washed twice with washing buffer, then blot dried. 50 xcexcl of the inhibitor solution diluted with assay buffer (2% DMSO final) is added to each well, followed by 50 xcexcl of Eu3+-VCAM-1 IgG diluted with assay buffer at 2 nM. The plate is incubated at room temperature for at least 30 min. Each well is then washed four times with washing buffer and blot dried. 100 xcexcl of DELFIA Enhancement solution is added to each well, followed by agitation of the plate at room temperature for 5 min. Fluorescence of each sample is then measured (e.g., DELFIA Fluorometer 1234, Wallace, Inc., USA). In this assay, the washing buffer comprises 25 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 4 mM MnCl2; the assay buffer comprises 25 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 4 mM MnCl2, 1% BSA, and 20 xcexcM DTPA.
The VLA4 inhibitors may be further characterized in in vivo assays. One such assay examines the inhibition of eosinophil infiltration into the bronchoalveolar lavage fluid in the mouse (murine) model. In this assay, the animals are treated with cyclophosphamide on day 0. On days 2 and 14, the animals are immunized intraperitoneally with Ascaris suum extract. Seven days later, the animals are treated with various doses of the VLA-4 inhibitor. Shortly after drug administration, the animals are challenged with Ascaris suum extract by instillation into the trachea. Bronchoalveolar lavage of the animal is performed by instilling saline into the lung, 48 hours later. Total cell and eosinophil counts in the lavage are determined.
In the murine model of Ascaris-induced bronchial inflammation, one of the representative compounds (example number 32) inhibited eosinophil infiltration by 49% at an oral dosage of 30 mg/kg. By contrast, a representative prior art compound, 4-(Nxe2x80x2-2-methylphenylurea)phenylacetyl-LDVP-OH, described in WO 97/03094, did not inhibit eosinophil infiltration (% inhibition=xe2x88x922%) at an oral dosage of 50 mg/kg.
Other representative compounds were also tested in mice. The dosage, route of administration and inhibitory effect of representative compounds (hereinafter, all tested compounds are referenced by compound number provided in the Synthetic Examples) are shown in Table 5.
The compounds of the present invention may also be further characterized in other in vivo assays, such as the eosioophil accumulation model tested in the rat. Fifty xcexcg of Compound 48/80 was injected into the pleural cavities of male Sprague Dawley rats. After 24 hrs, each cavity was washed twice with Hank""s Balanced Salt Solution containing 0.2% EDTA. Total cell and eosinophil counts were determined. Test compounds were given intraveneously, orally or subcutaneously, b.i.d. at 0 and 8 hours. The dosage, route of administration and inhibitory effect for the test compounds are shown in Table 6.
A compound was dissolved or suspended with an appropriate solvent at 1 mg/ml. Female Balb/c mice (7-9 weeks old) were given the compound orally. Blood samples were collected from the postcaval vein of the anesthetized mice after fifteen minutes. Serum was prepared and stored at xe2x88x9220xc2x0 C. Serum concentration of the compound was determined from inhibitory activities of the diluted serum by a direct binding assay using VLA-4-expressing cells and VCAM-IgG fusion protein. Serum concentration determined by this method correlated well with the concentration determined by LC/MS/MS methodologies. The dosage, route of administration and resulting inhibitory effect for the test compounds are shown in Table 7.
Pharmacokinetic parameters of exemplary compounds, in mouse, rat and monkey models, are shown in Tables 8, 9 and 10.
Pharmacokinetic parameters and the time course of the serum concentration of a single intravenous dosage (2 mg/kg) of a representative compound and ATENOLOL {4-[2xe2x80x2-hydroxy-3xe2x80x2-isopropylamino)proppyxy]phenylacetamide; Sigma Chemical Co., code no. A-7655}are summarized in Tables 10 and 11 for the monkey model.
Preparation of VCAM IgG Fusion Protein
A VCAM IgG fusion protein containing the one to seven immunoglobulin domains of VCAM-1 (D1D7) ligated to the hinge (H), CH2 and CH3 regions of human IgG1 was used in the binding assay.
Construction of a Stable Cell Line Expressing D1D7-VCAM IgG Fusion Protein
An Epstein-Barr virus based, episomal plasmid containing a D1D7-VCAM IgG fusion gene under transcriptional control of the CMV promoter, was transfected into 293E human embryonic kidney cells. Stably transfected cells were selected using 250 xcexcg/mL hygromycin in DMEM with 10% fetal calf serum. The cells secreted D1D7 VCAM IgG fusion protein into the medium cumulatively for up to 9 days.
Purification of D1D7 VCAM IgG Fusion Protein
The cells were cultured in DMEM with 10% fetal calf serum for 2 days, then changed to CCM5 medium and cultured for a further 10 days. The medium was centrifuged, filtered and then incubated overnight with Protein A Sepharose 4. The Protein A Sepharose was washed extensively and the D1D7 VCAM IgG fusion protein bound was eluted using 100 mM citric acid, pH 3.
Preparation of Europium Labeled-D1D7 VCAM IgG Fusion Protein
The D1D7-VCAM IgG fusion protein, at 1 mg/mL, was dialyzed against 50 mM NaHCO3, 0.9% NaCl, pH 8.5. The fusion protein was added to one vial of europium-labeling reagent (DELIA labeling kit from Wallac, Gaithersberg, Md.; catalog no. 1244-302) and incubated at room temperature in the dark overnight. The labeled protein was purified using a Sepharose G10 column and assayed for the europium content and protein concentration. The protein was stored at minus 80xc2x0 C. until used.
Construction of Cell Line Expressing VLA-4 (CHO/VLA-4)
A CHO cell line stably transfected with the cDNA of xcex14 and xcex21 was used in the binding assay. The gene for human xcex14 was obtained from the American Type Culture Collection and recloned between the XhoI and Xba sites of the mammalian expression vector pCI-neo Promega, Madison, Wis.). The xcex21 gene was amplified by PCR from human peripheral leukocyte cDNA and engineered such that the start codon was placed in the context of a consensus Kozak sequence. The gene was recloned into pCI-neo downstream of the CMV promoter and chimeric intron.
CHO-K1 cells were stably co-transfected with plasmids encoding the xcex14 and xcex21 genes, and single cells expressing high levels of VLA-4 were selected by fluorescence cell sorting (FACS). The antibodies used in FACS analysis were: anti-xcex14-PE conjugated (PharMingen, San Diego, Calif.) and anti-xcex21-FITC conjugated (Biosource, Camarillo, Calif.). A cell line 4B4, which expresses 400,000 and 300,000 sites/cell of the xcex14 and xcex21 subunit, respectively, was used in the binding assay. The subunit numbers were determined by FACS analysis, using Quantum Simply Cellular microbeads (Flow Cytometry Standards Corporation, Puerto Rico) as standards. The cells were maintained in F12 medium, containing 10% fetal bovine serum, 10 mM HEPES, pH 7.5, 0.5 mg/mL G418, using a 1:48 passage/week.
The CHO/VLA-4 cells were seeded in a 96-well microtiter plate at 30,000 cells/well and incubated at 37xc2x0 C., 5% CO2 for 48 hours until confluent. On the day of assay, the wells were emptied and washed twice with 350 xcexcl of a washing buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2 2 mM MnCl2. The plate was then drained and blotted dry on paper towels to remove buffer.
The test compound was serially diluted in assay buffer (washing buffer together with 0.1% bovine serum albumin, 20 xcexcM DTPA and 1% dimethysulfoxide), in the presence of 2 nM of europium-labeled D1D7-VCAM IgG fusion protein. Final concentrations used ranged from 0.1 mM-10 xcexcM. 50 xcexcl aliquot of the test compound mixture was added to duplicate wells in the plate. Control wells for total binding received no test compound. Non-specific binding wells contained an anti-xcex14 monoclonal antibody (L25.3, Becton Dickinson, Bedford, Mass.).
The cells were allowed to incubate with the test compound mixture, in the presence of europium-labeled D1D7-VCAM IgG fusion protein at room temperature for at least 30 minutes. The cells were then washed three times with 350 xcexcl of washing buffer, using a Skatron plate washer and blot dry. An 100 xcexcl aliquot of DELFIA Enhancement solution was added to each well, followed by gentle agitation at room temperature for 10 minutes. The amount of europium-labeled VCAM-IgG fusion protein bound was determined by time-resolved fluorescence measurement (Model: Victor(trademark), Wallac Inc., Gaithersberg, Md.).
Percent binding was calculated as: [(FTxe2x88x92FNS)xe2x88x92(FIxe2x88x92FNS)]/(FTxe2x88x92FNS)xc3x97100 wherein FT and FNS is the fluorescence signal of the europium labeled D1D7-VCAM IgG fusion protein bound to cells, in the absence of test compound and containing an anti-xcex14 monoclonal antibody, respectively. FI is the fluorescence in wells containing a test compound. The IC50 (concentration of the inhibitor to inhibit 50% binding of VACM to CHO/VLA-4 cell) was determined by a curve fitting routine, PRIZM (GrapbPad Software, Inc., San Diego, Calif.).
This secondary functional assay was used to determine the potency of a test compound in inhibiting VLA-4 mediated cell adhesion.
Preparation of VCAM Coated Plate
A 50 xcexcl aliquot of the D1D7-VCAM IgG fusion protein (1.5 xcexcg/mL in phosphate buffered saline, PBS) was added to each well of a 96-well Costar flat bottom plate (Costar, Franklin Lakes, N.J., catalog no. 2580). The plate was then incubated overnight at 4xc2x0 C. On the day of assay, the wells were emptied and washed twice with 350 xcexcl of PBS. The plate was then blocked with 100 xcexcl of 1% bovine serum albumin (BSA, Sigma, cat #A9418) in PBS at room temperature for at least a hour.
Cell Preparation
Jurkat cell (clone E6-1) was obtained from American Type Cultured Collection and was maintained in RPMI medium, 10 mM HEPES, pH 7.5, 1 mM sodium pyruvate, 10% FCS, using a 1:64 passage/week. Just prior to running the assay, Jurkat cells were labeled with 5 xcexcM of calcein-AM (Molecular Probe, Eugene, Oreg., catalog no. C1430) in RPMI medium, at room temperature for 30 min in the dark. Following labeling, cells were washed twice with RPMI medium and resupended at 1xc3x97106 cells/mL.
Cell Adhesion Assay
Immediately before the assay, the BSA solution was emptied from the VCAM-coated plate. The plate was then washed twice with RPMI medium. A 100 xcexcl aliquot of the labeled Jurkat cells was added to each well, followed by the addition of 50 xcexcl of the inhibitor solutions. Final inhibitor concentrations range from 1 nM to 10 xcexcM and each concentration was tested in triplicates. The inhibitor and cells were allowed to incubate at room temp for 1 hr in the dark. Following the incubation, the plate was immersed gently into a container filled with PBS, then inverted face down under PBS. The wells were drained and blotted dry on a layer of paper towel. A 50 xcexcl aliquot of 0.1% Triton X-100 was added to each well. The plate was incubated in the dark for 10 min. Adhesion of Jurkat cell was quantitated in a Millipore Cytofluor 2300 System plate reader set al 485 nM excitation and 530 nM emission. The IC50 (concentration of the inhibitor to inhibit 50% Jurkat cell adhesion) was determined by a curve fitting routine, PRIZM (GraphPad Software, Inc., San Diego, Calif.).
Compounds of the present invention may be prepared by standard chemical synthesis methods, as well as by methods of combinatorial chemistry, such as that described in Published PCT application, WO 95/30642.